CN104740625B - Load the preparation method of the LDH@SiO2 nanoparticles of F gene of NDV strain DNA - Google Patents
Load the preparation method of the LDH@SiO2 nanoparticles of F gene of NDV strain DNA Download PDFInfo
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Abstract
Load the LDH@SiO of F gene of NDV strain DNA2The preparation method of nanoparticle, it is related to a kind of preparation method for loading ewcastle disease DNA vaccination nanoparticle.It is to solve existing DNA vaccination antigen intramuscular administration easily to be degraded by the metabolic enzyme in intestines and stomach and sour environment, need to repeatedly it be immunized, sustained release and mucosa-immune can not be realized, Plasmid DNA immunization originality is weaker, the problem of could need to effectively inducing immune response with stronger immunologic adjuvant or suitable genes delivery system.Method：First, SiO is synthesized using sol-gal process2Nanoparticle；2nd, pVAX1 optiF/SiO2The synthesis of nanoparticle；3rd, ewcastle disease F gene plasmid DNALDH@SiO are carried2The synthesis of nanoparticle.Easily be degraded instant invention overcomes antigen DNA, the immune duration is short, often the shortcomings of, method is simple, mild condition, and production cost is low, is easy to large-scale production.For DNA vaccination field.
Description
Technical field
The present invention relates to a kind of preparation method for loading ewcastle disease DNA vaccination nanoparticle.
Background technology
As a kind of poultry communicable disease of acute high degree in contact --- ewcastle disease (ND), it is by NDV
(NDV) caused by, huge economic damage is caused in the range of world's aquaculture, is also had simultaneously for industry development huge
Potential threat.As can be seen here, newcastle disease vaccine development and effective prevention and control are of great immediate significance and higher to aviculture
Economic benefit.Relative to conventional vaccine, DNA vaccination has the advantages of uniqueness, is embodied in and is fundamentally solving reversion
Problem and gene mutation problem, and avirulence is replied, technique is easily realized, immune the duration is long, cost price is low, quality wind
Danger is smaller, at the same can excitating organism humoral immunity and cell immune response, the advantages that being easy to store and transport, have necessarily
Application practice and far-reaching Research Prospects.But current DNA vaccination is there is some shortcomings, as bioavilability is low, not up to thin
Just it is degraded before born of the same parents, the shortcomings of immune response is weak, in addition, its administering mode majority is in the form of intramuscular injection, for ewcastle disease
This kind of mucous membrane infectious disease caused by respiratory tract or alimentary canal, after intramuscular administration, it is difficult to which cell is passed through by diffusion way
Film, it is difficult to reach in the antigen presenting cells such as phagocyte, BMDC (APCs), therefore result in the amount of antigen given expression to
Low, immune effect is bad.Therefore, mucosa-immune has the function that very important in ewcastle disease preventing and treating.
The selection of gene delivery vector directly affects the effect of gene therapy.At present in gene therapy and gene vaccine
In research, most widely used gene delivery vector is viral vector.But Jesse's Ge Er Singh's events cause people for disease
The security of virus gene carrier generates suspection.Nano material has the security of non-virus carrier as gene delivery vector,
There is its unique advantage again.Two kinds of nano materials can be combined into one by the nano material of shell-core structure, so as to play each
The characteristics of material, and there is as gene delivery vector the security of non-virus carrier, DNA can be protected again not by nuclease
Degraded, strengthen its stability, extend the antigen presentation time, improve transfection efficiency etc..
The content of the invention
Easily dropped the present invention is to solve existing DNA vaccination antigen intramuscular administration by the metabolic enzyme in intestines and stomach and sour environment
Solution, need to repeatedly be immunized, it is impossible to realize sustained release and mucosa-immune, Plasmid DNA immunization originality is weaker, need to use stronger immunologic adjuvant
Or suitable genes delivery system the problem of could effectively inducing immune response, there is provided one kind load F gene of NDV strain matter
Grain DNA LDH@SiO2The preparation method of nanoparticle.
The technical scheme is that SiO is prepared by sol-gel method2Nanoparticle, then using 3- aminopropyls three
Ethoxysilane (APTES) modifies SiO2, make its surface modification into amino (APTES-SiO2), positively charged；By model drug
PVAX1-optiF and APTES-SiO2Connection, prepare pFDNA-SiO2-NPS；Using coprecipitation in pFDNA-SiO2-NPSSurface
Precipitate layered double-hydroxide (LDH), synthesis pFDNA-LDH SiO2-NPs。
The LDH@SiO of present invention load F gene of NDV strain DNA2The preparation method of nanoparticle, according to the following steps
Carry out：
First, SiO is synthesized using sol-gal process2Nanoparticle；
2nd, pVAX1-optiF/SiO2Nanoparticle (pFDNA-SiO2- NPs) synthesis
A、SiO2Amination：
Dose volume percentage concentration is that the ethanol of 1%~1.5% APTES (APTES) is molten
Liquid, weigh 1~1.5mg SiO2Nanoparticle is placed in above-mentioned solution, at room temperature 10~12h of magnetic agitation, and precipitation is collected by centrifugation,
Precipitation is washed with absolute ethyl alcohol three times, and precipitation is dried at room temperature, produces amidized SiO2Nanoparticle (APTES-SiO2)；
B, amidized SiO is weighed2Nanoparticle is scattered in PBS (pH7.4) cushioning liquid, is configured to 1mg/mL nanometer
Grain suspension, DNA pVAX1-optiF is added to nanoparticle suspension, is slowly inhaled with liquid-transfering gun and is played mixing, is incubated at room temperature
15min, 4 DEG C, 10000~11000r/min centrifugation 10min collection precipitations, is washed three times, precipitation PBS weight with PBS
It is outstanding, produce pFDNA-SiO2-NPs；
3rd, ewcastle disease F gene plasmid DNALDH@SiO are carried2Nanoparticle (pFDNA-LDH@SiO2- NPs) synthesis：
A, 6.3g NaOH and 9.1g NaNO are weighed3It is dissolved in the deionized water after 72mL boils, obtains solution A, by 16g
Mg(NO3)2·6H2O and 11.7g Al (NO3)3·9H2O is dissolved in the deionized water after 63mL boils, and obtains solution B；
B, 0.8g pFDNA-SiO are added into solution A2- NPs, in N2Under protection and stirring condition, by solution B with 3mL/
Min speed is slowly dropped in above-mentioned mixed liquor, continues that 1~1.5h is stirred at room temperature after being added dropwise；It is heavy to be collected by centrifugation
Form sediment, it is neutrality wash with the deionized water after boiling and be precipitated to supernatant, and room temperature in vacuo is drying to obtain pFDNA-LDH@SiO2-
NPs。
In step 2 of the present invention DNA pVAX1-optiF before the applying date in《Codon optimization strengthens new city
The expression and immune effect of epidemic disease virus DNA vaccine》(Chinese zoonosis journal, 2009,17 (2)：Disclosed in 8-16)
Deliver, can be obtained by author.
Nano-particle prepared by the present invention can protect DNA not degraded by DNA enzymatic, size tunable.
PFDNA-LDH@SiO prepared by the present invention2- NPs average grain diameters are 470.1nm, polydispersity coefficient 0.005,
Zeta potential is+16.82mV, and the envelop rate of nano-particle is more than 90%, and drugloading rate is more than 45%.
The technical scheme is that the mesoporous SiO of different-grain diameter is synthesized using solution-gel method2, then existed with coprecipitation
Mesoporous SiO2Surface coating LDH, form LDH@SiO2, complete to take F gene of NDV strain DNA by nuclear structure
Carry, establish safe level mucosa-immune delivery system (the pDNA-LDH@SiO of ewcastle disease DNA vaccination nanosizing2-NPs).The present invention
The LDH@SiO of synthesis2Both there is good dispersion in LDH bodies, sensitive to pH, Zeta potential is high, have again can extend antigen be detained,
Beneficial to DC identifications, ripe and to lymph node chemotactic the advantages of promotion DC, solve simple LDH and asked as what DNA vaccination was faced
Topic；Meanwhile LDH@SiO2With SiO2Meso-hole structure feature, breach LDH and carry the limited bottleneck of DNA.
PFDNA-LDH@SiO prepared by the present invention2- NPs overcomes antigen DNA and is easily degraded, the duration is immunized
Short, the shortcomings of immune time is more, and preparation method is simple and easy, mild condition, production cost is relatively low, is easy to large-scale production.
Brief description of the drawings
Fig. 1 is the transmission electron microscope figure of silica prepared by embodiment 1；
Fig. 2 is pFDNA-SiO prepared by embodiment 12- NPs transmission electron microscope figure；
Fig. 3 is pFDNA-SiO prepared by embodiment 12The agarose gel electrophoresis figure of the anti-DNA enzymatic degradeds of-NPs
Fig. 4 is pFDNA-LDH@SiO prepared by embodiment 12- NPs infrared spectrogram；
Fig. 5 is pFDNA-LDH@SiO prepared by embodiment 12- NPs XRD；
Fig. 6 is pFDNA-LDH@SiO prepared by embodiment 12- NPs external release profile；
Fig. 7 is pFDNA-LDH@SiO prepared by embodiment 12- NPs transmission electron microscope picture；
Fig. 8 is pFDNA-LDH@SiO prepared by embodiment 12- NPs grain size distribution.
Embodiment
Technical solution of the present invention is not limited to act embodiment set forth below, in addition between each embodiment
Any combination.
Embodiment one：Present embodiment loads the LDH@SiO of F gene of NDV strain DNA2Nanoparticle
Preparation method, carry out according to the following steps：
First, SiO is synthesized using sol-gal process2Nanoparticle；
2nd, pVAX1-optiF/SiO2Nanoparticle (pFDNA-SiO2- NPs) synthesis
A、SiO2Amination：
Dose volume percentage concentration is that the ethanol of 1%~1.5% APTES (APTES) is molten
Liquid, weigh 1~1.5mg SiO2Nanoparticle is placed in above-mentioned solution, at room temperature 10~12h of magnetic agitation, and precipitation is collected by centrifugation,
Precipitation is washed with absolute ethyl alcohol three times, and precipitation is dried at room temperature, produces amidized SiO2Nanoparticle (APTES-SiO2)；
B, amidized SiO is weighed2Nanoparticle is scattered in PBS (pH7.4) cushioning liquid, is configured to 1mg/mL nanometer
Grain suspension, DNA pVAX1-optiF is added to nanoparticle suspension, is slowly inhaled with liquid-transfering gun and is played mixing, is incubated at room temperature
15min, 4 DEG C, 10000~11000r/min centrifugation 10min collection precipitations, is washed three times, precipitation PBS weight with PBS
It is outstanding, produce pFDNA-SiO2-NPs；
3rd, ewcastle disease F gene plasmid DNALDH@SiO are carried2Nanoparticle (pFDNA-LDH@SiO2- NPs) synthesis：
A, 6.3g NaOH and 9.1g NaNO are weighed3It is dissolved in the deionized water after 72mL boils, obtains solution A, by 16g
Mg(NO3)2·6H2O and 11.7g Al (NO3)3·9H2O is dissolved in the deionized water after 63mL boils, and obtains solution B；
B, 0.8g pFDNA-SiO are added into solution A2- NPs, in N2Under protection and stirring condition, by solution B with 3mL/
Min speed is slowly dropped in above-mentioned mixed liquor, continues that 1~1.5h is stirred at room temperature after being added dropwise；It is heavy to be collected by centrifugation
Form sediment, it is neutrality wash with the deionized water after boiling and be precipitated to supernatant, and room temperature in vacuo is drying to obtain pFDNA-LDH@SiO2-
NPs。
Using sol-gal process synthesis SiO in present embodiment step 12Nanoparticle concretely comprises the following steps：
90mL absolute ethyl alcohols, 12.5mL deionized waters, 2mL concentrated ammonia liquors (NH are added into the beaker of cleaning successively3Content is
28%), and 200r/min magnetic agitations make it well mixed at room temperature；Afterwards, under magnetic stirring, disposably quickly to it
Middle addition 10mL TEOS, solution gradually becomes milky after adding TEOS, continues magnetic agitation 2h at room temperature；Reaction stops
Afterwards, 4 DEG C, 10000r/min SiO is collected by centrifugation2Nanoparticle, absolute ethyl alcohol wash precipitation and precipitation are placed in into air afterwards three times repeatedly
Middle drying, that is, obtain SiO2Nanoparticle.
Tetraethyl orthosilicate used in present embodiment, concentrated ammonia liquor are all common chemical reagent, and production cost is low, suitably
Large-scale production.SiO prepared by the present invention2Surface is smooth, form is regular, spherical in shape, particle size is homogeneous, good dispersion, nothing
Phenomena such as being substantially adhered, collapsing
The appearance of novel nano-material, possibility is provided to obtain the DNA vaccine vector of high-quality.Layered bi-metal hydrogen-oxygen
Compound (LDHs) is a kind of layer post being made up of positively charged layers of metal hydroxides and interlayer filling exchangeable anions
Shape compound.LDH has lot of advantages as gene delivery vector：1) DNA classes large biological molecule can be stable in the presence of LDH nanometers
In composite；2) internal good dispersion, in pH 3-5 environment, rate of releasing drug is fast；3) biocompatibility and slowly releasing effect are good,
And have higher Zeta potential, easily contacted with cell surface, into cell interior, improve transfection efficiency.But LDH is as gene delivery
There is also some shortcomings for carrier：1) LDH interfloor distances are smaller, and DNA is difficult insertion；Though 2) DNA is adsorbed in LDH surfaces
It can so complete to carry, but LDH can be made to weaken DNA protective effects, and DNA lift-launch amounts are very limited.With mesoporous SiO2For original
The nano material of material has the characteristics that：1) there is high specific pore volume, more DNA macromoleculars can be accommodated；2) it is rich in silicon hydroxyl
The surface of base, it is easy to accomplish functionalization；3) there is pH sensitiveness, can be by adjusting release of the pH realizations to DNA；4) it is unique
Hole wall structure can provide protection for DNA.Because two kinds of nano materials can be combined into one by the nano material of shell-core structure,
The characteristics of playing respective material.
Embodiment two：Present embodiment is unlike embodiment one：Dose volume hundred in step 2 A
Divide the ethanol solution for the APTES that concentration is 1%.It is other identical with embodiment one.
Embodiment three：Present embodiment is unlike embodiment one or two：Weighed in step 2 A
1mg SiO2Nanoparticle is placed in the ethanol solution of APTES.Other and embodiment one or two
It is identical.
Embodiment four：Unlike one of present embodiment and embodiment one to three：Matter in step 2 B
Grain DNA pVAX1-optiF and amidized SiO2The mass ratio of nanoparticle is 1:5.It is other with embodiment one to three it
One is identical.
Embodiment five：Unlike one of present embodiment and embodiment one to four：4 in step 2 B
DEG C, 10000r/min centrifugation 10min collect precipitation.It is other identical with one of embodiment one to four.
Embodiment six：Unlike one of present embodiment and embodiment one to five：Dripped in step 3 b
Add and after finishing continue that 1.5h is stirred at room temperature.It is other identical with one of embodiment one to five.
Embodiment：
The present embodiment loads the LDH@SiO of F gene of NDV strain DNA2The preparation method of nanoparticle, by following step
It is rapid to carry out：
First, SiO is synthesized using sol-gal process2Nanoparticle：Added successively into the beaker of cleaning 90mL absolute ethyl alcohols,
12.5mL deionized waters, 2mL concentrated ammonia liquors (NH3Content is that 28%), and 200r/min magnetic agitations make its mixing equal at room temperature
It is even；Afterwards, under magnetic stirring, solution gradually becomes milky white after disposable quick addition 10mL TEOS thereto, addition TEOS
Color, continue magnetic agitation 2h at room temperature；Reaction stop after, 4 DEG C, 10000r/min SiO is collected by centrifugation2Nanoparticle, anhydrous second
Alcohol washs precipitation and precipitation is placed in into air drying afterwards three times repeatedly, that is, obtains SiO2Nanoparticle.
Transmission electron microscope figure such as Fig. 1 of silica prepared by the method, it is seen that the SiO of preparation2Surface is smooth, shape
State is regular, spherical in shape, particle size is homogeneous, good dispersion, without phenomena such as being substantially adhered, collapsing.
2nd, pVAX1-optiF/SiO2Nanoparticle (pFDNA-SiO2- NPs) synthesis
A、SiO2Amination：
Dose volume percentage concentration is the ethanol solution of 1% APTES (APTES), is weighed
1mg SiO2Nanoparticle is placed in above-mentioned solution, at room temperature magnetic agitation 12h, and precipitation is collected by centrifugation, and it is heavy to be washed with absolute ethyl alcohol
Form sediment three times, precipitation is dried at room temperature, produces amidized SiO2Nanoparticle (APTES-SiO2)；
B, amidized SiO is weighed2Nanoparticle is scattered in PBS (pH7.4) cushioning liquid, is configured to 1mg/mL nanometer
Grain suspension, DNA pVAX1-optiF is added to nanoparticle suspension, is slowly inhaled with liquid-transfering gun and is played mixing, is incubated at room temperature
15min, 4 DEG C, 10000r/min centrifugation 10min collection precipitations, is washed three times, precipitation is resuspended with PBS, is produced with PBS
pFDNA-SiO2-NPs.DNA pVAX1-optiF and amidized SiO2The mass ratio of nanoparticle is 1:5.
The pFDNA-SiO of preparation2- NPs transmission electron microscope figure such as Fig. 2.
3rd, ewcastle disease F gene plasmid DNALDH@SiO are carried2Nanoparticle (pFDNA-LDH@SiO2- NPs) synthesis：
A, 6.3g NaOH and 9.1g NaNO are weighed3It is dissolved in the deionized water after 72mL boils, obtains solution A, by 16g
Mg(NO3)2·6H2O and 11.7g Al (NO3)3·9H2O is dissolved in the deionized water after 63mL boils, and obtains solution B；
B, 0.8g pFDNA-SiO are added into solution A2- NPs, in N2Under protection and stirring condition, by solution B with 3mL/
Min speed is slowly dropped in above-mentioned mixed liquor, continues that 1.5h is stirred at room temperature after being added dropwise；Precipitation is collected by centrifugation,
It is neutral to be washed with the deionized water after boiling and be precipitated to supernatant, and room temperature in vacuo is drying to obtain pFDNA-LDH@SiO2-NPs。
Fig. 3 is pFDNA-SiO manufactured in the present embodiment2The agarose gel electrophoresis figure of the anti-DNA enzymatic degradeds of-NPs, it is seen that
SiO2DNA can be protected not degraded by DNA enzymatic；M swimming lanes are Maker, and 1 swimming lane is adds the DNA of DNA enzymatic, 2 swimming lanes
For DNA, 3-8 swimming lanes are SiO2The mass ratio of nano-particle and DNA is 1 respectively:5,1:1,5:1,10:1,20:1,
30:1。
Fig. 4 is pFDNA-LDH@SiO manufactured in the present embodiment2- NPs infrared spectrogram, it is seen that LDH@SiO2Characteristic peak
Fig. 5 is pFDNA-LDH@SiO manufactured in the present embodiment2- NPs XRD, it is seen that LDH is in cladding pFDNA-SiO2-
During NPs, crystal formation and structure all do not change, and the LDH crystalline phases of synthesis are single, better crystallinity degree.
Fig. 6 is pFDNA-LDH@SiO manufactured in the present embodiment2- NPs external release profile, it is seen that pFDNA-LDH@SiO2-
NPs has obvious slow release characteristics.
Fig. 7 is pFDNA-LDH@SiO manufactured in the present embodiment2- NPs transmission electron microscope picture, the pFDNA-LDH@of preparation
SiO2- NPs forms are very regular, and profile is complete, and without obvious adhesion, homogeneity is good, LDH and pFDNA-SiO2- NPs can
Relatively effective cladding is formed, is coated on pFDNA-SiO2The LDH of-NPs outer layers is without obvious laminated structure, but with Mi Dui's
Mode is coated on its surface.
Fig. 8 is pFDNA-LDH@SiO manufactured in the present embodiment2- NPs grain size distribution, it is seen that the pFDNA- of preparation
LDH@SiO2- NPs average grain diameters are 470.1nm.
Table 1 is the pFDNA-LDH@SiO prepared under the method2Caused IgG antibody potency after SPF chickens is immunized in-NPs, can
See the pFDNA-LDH@SiO of preparation2- NPs can effectively stimulate chicken body to produce higher mucosal immune response.
Table 2 is the pFDNA-LDH@SiO prepared under the method2Caused IgA antibody potency after SPF chickens is immunized in-NPs, can
See the pFDNA-LDH@SiO of preparation2- NPs can effectively stimulate chicken body to produce higher mucosal immune response.
IgG antibody potency (unit in the Post-immunisation serum of table 1：Log2)
Note：Data withRepresent, the upper right corner shows that group difference is notable without identical lowercase in same column data
(p ＜ 0.05).
IgA antibody potency (unit in the Post-immunisation serum of table 2：pg/mL)
Note：Data withRepresent, the upper right corner shows that group difference shows without identical lowercase in same column data
Write (p ＜ 0.05).
Claims (1)
1. load the LDH@SiO of F gene of NDV strain DNA2The preparation method of nanoparticle, it is characterised in that this method is pressed
Following steps are carried out：
First, SiO is synthesized using sol-gal process2Nanoparticle：90mL absolute ethyl alcohols, 12.5mL are added into the beaker of cleaning successively
Deionized water, 2mL concentrated ammonia liquors, and 200r/min magnetic agitations make it well mixed at room temperature；Afterwards, under magnetic stirring,
10mL TEOS are disposably added thereto, and solution gradually becomes milky after adding TEOS, continues magnetic agitation at room temperature
2h；Reaction stop after, 4 DEG C, 10000r/min SiO is collected by centrifugation2Nanoparticle, absolute ethyl alcohol washs precipitation repeatedly three times afterwards will be heavy
Shallow lake is placed in air drying, that is, obtains SiO2Nanoparticle；NH in the concentrated ammonia liquor3Content is 28%；
2nd, pVAX1-optiF/SiO2The synthesis of nanoparticle
A、SiO2Amination：
Dose volume percentage concentration is the ethanol solution of 1% APTES, weighs 1mg SiO2Nanoparticle
It is placed in above-mentioned solution, at room temperature magnetic agitation 12h, precipitation is collected by centrifugation, precipitation is washed three times with absolute ethyl alcohol, by mud chamber
The lower drying of temperature, produces amidized SiO2Nanoparticle；
B, amidized SiO is weighed2Nanoparticle is scattered in PBS cushioning liquid, is configured to 1mg/mL nanoparticle suspension, Xiang Na
Grain of rice suspension adds DNA pVAX1-optiF, is inhaled with liquid-transfering gun and plays mixing, is incubated 15min, 4 DEG C, 10000r/ at room temperature
Min centrifugations 10min collects precipitation, is washed three times with PBS, precipitation is resuspended with PBS, produces pFDNA-SiO2-NPs；Matter
Grain DNA pVAX1-optiF and amidized SiO2The mass ratio of nanoparticle is 1:5；
3rd, ewcastle disease F gene plasmid DNA LDH@SiO are carried2The synthesis of nanoparticle：
A, 6.3g NaOH and 9.1g NaNO are weighed3It is dissolved in the deionized water after 72mL boils, obtains solution A, by 16g Mg
(NO3)2·6H2O and 11.7g Al (NO3)3·9H2O is dissolved in the deionized water after 63mL boils, and obtains solution B；
B, 0.8g pFDNA-SiO are added into solution A2- NPs, in N2Under protection and stirring condition, by solution B with 3mL/min's
Speed is slowly dropped in above-mentioned mixed liquor, continues that 1.5h is stirred at room temperature after being added dropwise；Precipitation is collected by centrifugation, with boiling
Deionized water washing afterwards is precipitated to supernatant and is drying to obtain pFDNA-LDH@SiO for neutrality, room temperature in vacuo2-NPs。
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| CN104189922A (en) * | 2014-09-19 | 2014-12-10 | 黑龙江大学 | Preparation method of ND (New Castle-disease) DNA vaccine Ag@SiO2 carried nanoparticles |
| CN104310408A (en) * | 2014-09-30 | 2015-01-28 | 黑龙江大学 | Synthetic method of LDH@SiO2 shell-nuclear nano composite material |
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| CN104310408A (en) * | 2014-09-30 | 2015-01-28 | 黑龙江大学 | Synthetic method of LDH@SiO2 shell-nuclear nano composite material |
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